The present invention relates to methods, reagents and kits for the determination (qualitatively or quantitatively) of the presence of target nucleotide sequences, and particularly for determining the presence of target nucleotide sequences such as the structural genes for globin proteins or blood factor proteins where such sequences in double-stranded nucleic acid strands contain one or more restriction endonuclease cleavage sites.
Nucleic acid detection assays of various types have been documented in the technical and patent literature. These types of assays, and particularly those requiring detection of polynucleotides, in large part are based on the purine-pyrimidine base pairing properties of complementary nucleic acid strands in DNA-DNA or DNA-RNA duplices where DNA refers to deoxyribonucleic acid and RNA refers to ribonucleic acid. This base pairing process most frequently occurs through formation of hydrogen bonds in the pairing of adenosine-thymine (A-T) and guanosine-cytosine (G-C) bases in double-stranded DNA; adenosine-uracil base pairs may additionally be formed by hydrogen bonding in DNA-RNA hybrid molecules, as may A-T and G-C pairs. Base pairing of nucleic acid strands for determination of the presence or absence of a given nucleotide sequence typically occurs between sample nucleotide sequences and a probe polynucleotide (referred to hereinafter also as a probe nucleotide sequence) and is commonly termed nucleic acid hybridization or simply hybridization.
Frequently (as in U.S. Pat. No. 358,535 issued to Falkow et al. (1982)), nucleic acid hybridization assays require the sample nucleotide sequences to be immobilized to or on a solid matrix or substrate and to be rendered single-stranded if not already present in that form. The immobilized sample nucleotide sequences are then washed extensively before exposure to labeled probe polynucleotides. A specific application of nucleic acid hybridization technology of this type is described in U.S. Pat. No. 4,395,486 issued to Wilson et al. (1983) which proposes an assay limited to the detection of the rare structural gene for hemoglobin S. The assay described therein first obtains double-stranded sample or analyte DNA in a native double-stranded form and then subjects the contents of the reaction mixture to enzymatic cleavage by the restriction endonuclease Dde I. Restriction fragments including unique fragments of 175 and 201 nucleotide base pairs in length arise from a gene for hemoglobin A or including a unique fragment of 376 nucleotide base pairs in length arise from a gene for hemoglobin S. Thus the genotypes for normal (AA) individuals and for homozygous individuals with sickle cell disease (SS) can each be detected by physical analysis of the restriction endonuclease cleavage products by such methods as gel electrophoresis of the restricted DNA and subsequent detection of nucleotide sequences containing the appropriate portions of a hemoglobin gene by the blotting method of E. M. Southern (Journal of Molecular Biology vol. 98, pp. 503-517 (1975)). The presence of all three fragments in Southern blots is indicative of the heterozygous sickle cell trait (i.e., the AS genotype).
A group of inventors including some of the present inventors have described a polynucleotide sequence hybridization assay involving polynucleotide strand displacement in the copending application U.S. Ser. No. 607,885, filed May 7, 1984. Such an assay is based upon the selective displacement of a labeled polynucleotide from a complementary probe nucleotide sequence by an appropriate target nucleotide sequence (if present) and subsequent detection of the displaced labeled polynucleotide. Nucleic acid detection assays of this type with hybridization rate modifiers present during the nucleic acid strand displacement step of the assay are described in applications U.S. Ser. No. 684,305 and 684,308, each filed Dec. 20, 1984, and authored by the same aforementioned group of inventors. These assay methods each have limitations in determining the presence of target nucleotide sequences with single purine or pyrimidine base changes as compared to reference target nucleotide sequences.
A group of inventors including some of the present inventors have separately described in application U.S. Ser. No. 652,218, filed Sept. 19, 1984, a nucleic acid diagnostic assay based upon hybridization of analyte DNA to a labeled probe polydeoxynucleotide, cleavage of the product double-stranded DNA hybrid at a restriction site formed upon DNA duplex formation and detection of any cleaved and labeled double-stranded DNA fragments. While this method can detect certain single base changes at specified sites in analyte DNA strands, it has the potential drawback of an appropriate restriction site forming non-specifically at the half-restriction endonuclease recognition site of the labeled probe polydeoxynucleotide, leading to false positive results in the assay. Such a drawback may be reduced or eliminated by selecting conditions whereby productive (i.e., stable) nucleic acid hybridization events occur only when reasonably long tracts of base pairing (e.g., greater than about 10 base pairs) are allowed prior to the next step of the assay; such conditions may, however, impose certain limiting conditions on the procedure used in the assay.